Grant title for the next 5 years: Abstract: The theme of this award since its incejatipn In 1997 hias been to ghderstand how phosphorylation regulatds Opioid receptor slgnialirig. Iri'prior cycles, we defined sites bf opioid reclieptor phdsphoryl^itlon medisijting homologous, G-proteih receptor kinase 7. p-arrestin- dependehtr^ce|>torc|e8ensiti2^ip^^ In this rnu,opioid receptor and,pi[3;p^ signaling) V that;|Mntrol|edv jof;opioid;-receptof .;sigiriallngi;The,ii^phy^jologieai significance bf^tHMestyrosii:^ ph^fjllb^^ was est^Ilshed lay derhonstrailrig 4h(&lr ^i^qlejh #glJlatibri%plpid jSigjialiri^ [unreadable] :we!;prbp[unreadable]is6 to ruridi^rstifhd'^hic^ rdgtilat^i'bS;'Juri-^ klnase;phosiphpiylitlbn'e>^^ kapipi[unreadable] ppibld'r^^^pris^fdrmv slgnalirtg^cdilfvplexes:/^;Wi/^^ this previously. undiscp.verBd medris .pf.xe!ceptpri^^||,u^^ the .prpposed.flims: we: wpujd i) Id^nt^ thenSp^lhislril Sl^l^^ OfJgri,Kinase in tranisifeqteiififeK cells ieadihg from,opioid^feceptbr'actiy^tton.: to 4un Kin|ise.ijhbsphprylatibii Will be defined. 2J'We propose that theiopjoid receptor sld0e mecromplecular structure containing a jHlK-sensitive. cqmppnent reiq&li^ed fiprr^efnGi^ tb. the .effector. We will affinity purify the signaling cOmp|exahd.;identi|/the e^^ analysiSv This willbe usedvto ideiritiiythe'VfJK^h^^^^^ siilistrate withip the opldid receptor compiex. 3) The roles of specific cendldates'iden^^^^ aim #2 will be 'yalidated by targeted deletloFi and in wYw reboHstitutlpn'i^xp^^^^ the resulting bipchemiciiija^^;p^ibl6glcal:;.descHptIpns pf the opioid receptor signaling contiplex-Wiliprpvldei deeper Linder^^^^ Specific. Aim #1 Morphine activation of the triu opioid receptor and norBNI activation ofthe kappa opioid receptor both result In Jun N-temiinal Kinase (JNK) phosphorylation, which subsequently causes oplold-receptor Ihactlvation. For MOR, this takes the forni of acute analgesic tolerance to morphine and for KOR, thls'Fesults In long-lasting antagon|srh. FOiir different .Intemnediate kinases have been Implicated In JNK'activatioii: ASKI, TAK1. MLK.- and. M6KK1. Using MOR-QFP and KOR-GFP expressing HEK293 celjs, we will use-dominant negative forms pf these potential mediators to Identify the Idnase'responsible! Next, morphin'e-lli(e opioids Induce JNK activation, but not GRK/anrestiri- dependent intemalization, whereas high efficacy opioids lli<e fentanyl Ihdufce both. The role of InternaliSiiation Ih preventing JNK-mediated inaottyatlon Wl|j be assessed by using 0RK3 knockout mice . to prevent fentanyi-lnduced'Internalization of MOR. the hypothesis Is that p-iarrestln assbciatioh with 'IVIOR protects .tlie receptor complex ffon^ dNK-lrlduced inactivation. In addition, three different JNK 'Isofonns cah be.detected in HEK.andspinal cordtlssua.:Pilotdata su^gestthat JNKI Is preferentially 'activated by morphine. .Thls^III be confirmed by quantitative .analysts because, a preferentlai- . association suggests that JNK1. may be specifically associated with the MOR signaling oonriplex. The goal Of this ajmls to define the.steps connecting lyiOR and KOR actlvatlpn p JNK phosp'horyiatlon. . Specific AfmUl We have-begun to develop Immunorlsolatlon methods necessary tp purify MOR and KOR signaling complexes froril H^K and mouse.spinal cord using antl-i\40R and KOR antibodies . deVieloped'eaflief iri this award;We are titso'using the powefful Tandem Afflnity'PuriflcatkMi'prd^edur^ for receptor purification, these procedures have been described (Puig 2001;BUrckstOmmer 2006^, Daula.t 2.007)( 'Local expertise Is readily available (Angers 2006;tyssand 2008), .and Ning Zheng,iCliri8 . [unreadable] HS^ue and Randy MoOn (faculty members In .the UW Department of Pharmacology) have offered to help me purify, the opioid signaling complexes and Iiiterpret the mass spectrosddplc.data using this technique. We have generated adeno-associated viral. (AAV) constmct^ expressing the streptavidin -. binding-peptide Ijnked to the calmodulin binding peptide than linked to the Nrterminus of either KOR or ' MOR.;'.these Ay[unreadable]S/ vectors will be Injected intp_mouae brain or expressed In HEK cells. Following the protocol described by .daulat (2007), membrane pnsteln will t>e detefgent S.oIubilIzed, adsorbed to Streptavidin resin, then calmodulin resin: The eluted proteins wiilbe digested thei^ resolved t)y 2D niass sjsectroscopy In the UW Proteomics facility. Differences in Isolates from tissues pretreated W.Ith either 'morplilne,'norBNj, fentanyl or U50,488 iand blocked by JNK.Iiiihibitor will be used to Identify critical JNK- suljstrate candidates. ^ :, . . Specific Alth #3 In addition to the discovery based proteomlcapprdach described In aim 2;we will also use a hypothesis driven approach to .assess the role pf predicted candidates. Two plausible JNK targets -are 1) JNK-^hduced ubiquitination of G-alpha.(proteins, and 2) .RGS9.. the former will be assessed.'by I'mmunopreclptitatlon of Gd and Gao and western blotting 43y anti-ubiquit[n antibodies. darz6n has shown that activation.of the .MOR by morphine, results In Grprbteln-uncoupllngelnd sequestration by RGS proteins (Oarz6n 2664 and 2005, Rodrlguez-Munoz 2007y.,Furthenifi'Ore,.RG$ - proteins that are.khdwn to co-localize dnd co-pracipltate with the MOR, such as RGS9 (Zacharlou "2003,..Qarz6n 2005), posses'the archetypical JNK phosphorylation'dpriialh..In'their amino acid . secjUences.. This suggests RGS proteins 0s a potential mechanism fpr morphine-induced receptor desensitization and tolerance, but comparison studies with other MOF^-^elective agonists .^re.lacking. To address the role of JNK In the morphine-induced RGS effects, animals will be-pretreated with. SP600;) 25;(J>iK-l), and we will nfieasure (aa sequestration and RGS actJvafidri following adinihistration - 'of morphine or fentaiiyl using colmmunopreplpitatlon and western blot techniques. Phosphorylatioh-of RGSd will'be measured by precipitating R6S9-2 using antibodies specific'for .the N-tenninu$ (Santa Cruz Biotechnologies) and probing with an antibody generated ag'dlnst .generalphosphorylated serine/threonine reisldues (BD Biosolences). Secjues^atlpn pf Ga subunitsi will be measured by coimmunoprecipitation of F^GSd using IgG antibodies against Ga as a probe;as described (Garz6n. 200S)..lf JNK activation Is required for tiiese evl^nfs. It Is expected thatpretreatmeht with SP600125.will prevent ,RGS phosphorylation and Ga coimrhuiiopreclpltation in tissue from animals treated with'. [unreadable] morphine but noil fentanyl. Streptavldlh figure 1: Scheme showing a proposed Calmodulin binding domains cphfiguratloh of the tandem affinity mu opioid receptor complex. We propoise that a JNK substrate Is a vital component of thereceptor signaling complex that becomes inactivated following phosphorylation. -. Dflce 'phosphdiylatadrthe JN|^ substrate^occludesth ,Ga binding idornain of the refeeptor ahcl iiidciis signaling. For kc>i^) this .assqclatlbh. Is'Stable JNK;and pSBuap-lrreyersIble;'for Mdt^ this scibstrate'" assoclatibn p^'rsi^t^forjess thah'6^^^ for that difference Will become;blearer once.the structures of tlie JNK substrates are determined. JNK. [unreadable]fSceS-' Angers S, LI T, YI X, MacCdss MJ, Moon RT, Zheng N. (2006) Molecular architecture ahd assembly of 'tKe. DpB1-c:UL4AUbiqulOniigas6 machinery. W^^^ BQrCkstUmhier^T.KBenriett KL, Pheradovic A, SchOto G, Hantschel 0, Superti-Furga 6, Bauch A. '(2006) Ah efficlerittandern affinity purif^^ procedure for Interaction proteomics In mammalian ,cells.;Klat;Mel;h6dSv3('i2):1p13-S/^ Daulati^-AM;Mauri(iyen"|;P P., Guiilaume, JL., Broussard, C, Monsan^t, Bj, Delagcahg'e, P., Jockerai'Ft. (2067) Puri^ and Identification of GProtelncoupled Receptor Protein Cortiplexes undetNatlve.'GQnditI GarzPh j;Rpdrigf^ez-Mufloz^^ iyiorphine alters the selective association : betweeh-mu-6pIoid receptors and specific RGS proteins In mOuse perlaquediictal gray matter. NeuropheirTn 48: fi53 Gar76n J, Fiodrtgijez-Mufloz M, L6pez-Fando A, Sinchez-Blizquez P. 2005. Activation of |i-oplold Receptdrs trahsferis Control of Ga Subunits to the Regulator of G-prOtelh Signaling RGS9-2. JBC 286(iO):B95l LyssandJS;DeFino MC, Tang XBi Hertz.AL;Feller DB, Wacker JL, Adams ME, Hague C. (2008) BIbbd pressure is regulated by an alphal D-adrenergic receptor/dystrophih signalosome. J Biol C/jem. 283i[27).'18792-i800. ^ Puig 0i,Caspary P,'iRlgaUt:.G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Si^raphln B^ (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. 224(3):218-29. Rodrlguei-Muftoz M, BermUdez D, S^nchez-Bldzquez P, Garz6n J. 2007. Suriioylated RGSTRZ Proteins Act as Scaffolds for Mu-Oploid Receptors and G-Protein Complexes In Mouse Brain. Neuropsychopharm 32:842 Zacharlou V, (Seorgescu D, Sanchez N, Rahman Z, DILeone R, Berton O, Neve RL, Sim-Selley LJ, Seiley DE, Gold SJ, Nestler EJ. 2003. Essential role for RGS9 in opiate action. PAMS 100(23): 13656